- Email: [email protected]
Cell envelope structure of the psychrophilic bacterium micrococcus cryophilus: A scanning electron microscope study
Micron and Microscopica Aria, Vol. 16, No. I, Pp. 5-16, 1985. Printed in Great Britain.
0739-6260/85 13.00+0.00 ~ 1985 Pergamon Press Ltd.
CELL ENVELOPE STRUCTURE OF THE PSYCHROPHILIC BACTERIUM MICROCOCCUS CRYOPHILUS: A SCANNING ELECTRON MICROSCOPE STUDY GEOFFREY
M. LLOYD,*t IOLO
AP GWYNN~and NICHOLAS
J.
RUSSELI]’~
*Department of Biochemistry, University College, Cathays Park, P.O. Box 78, Cardiff, CFI 1XL, U.K. and ~Department of Zoology, University College of Wales, Penglais, Aberystwyth, Dyfed, SY23 3DA, U.K. (Received 3 October 1984)
Abstrac*—The surface morphology and structure of the cell envelope and associated capsule of the Gramnegative psychrophilic bacterium Micrococcus cryophi/us have been investigated by scanning electron microscopy, following treatment of cells with NaCI, EDTA, sucrose, Tris-HCI and phosphate buffer. These treatments removed varying amounts ofthe extracellular capsule and damaged the outer membrane to differing extents. EDTA, and to a lesserdegree Tris-HCI, caused most damage, but compared with E. co/i relatively less damage occurs, probably due to the presence of the capsule in M. cryophilus. Besides having a protective role, it is proposed that the capsule associates with the outer membrane by ionic interactions, and probably increases intercellular interaction and cell contact with the substratum. We confirm that the outer membrane has a wavy profile, and show that it is thrown into folds whose depth and regularity depend on whether Tris—HCI or phosphate buffer is used. The troughs ofthese outer membrane folds tend to retain capsular material unless cells are washed with NaCl, and the peaks of the folds form vesicles which can bleb from the surface; blebbing is greatly increased by EDTA. Index keywords: Micrococcus cryophilus, Gram-negative bacteria, cell envelope structure, psychrophilic bacteria, bacterial capsule, scanning electron microscopy.
INTRODUCTION Micrococcus cryophilus is a psychrophilic bacterium, isolated by McLean et al. (1951) from sausages made with chilled meat. Although classified originally as a Gram-positive or Gram-variable coccus, subsequent transmission electron microscopy and freeze-fracture investigations have shown it is a Gram-negative organism (Mazanec et a!., 1966; Sleytr and Kocur, 1971). Biochemical confirmation has come from studies of the guanosine and cytosine content of its DNA (Bohã~eket a!., 1967). the type of peptidoglycan in its cell wall (Kandler et a!., 1970), and from the content and composition of its lipid (Russell, 1974). Micrococcus cryophilus has a typical
Gram-negative cell wall, consisting of a cytoplasmic (‘inner’) membrane, a peptidoglycan layer, and outside that a lipopolysaccharidecontaining outer membrane. In addition there is a capsule external to the outer membrane (Mazanec et al., 1966). The cytoplasmic and outer membranes have been separated and purified, and their compositions compared (Lloyd and Russell, 1984). This purification procedure involved converting intact bacteria to sphaeroplasts using EDTA and lysozyme, Tris—HC1 buffer and sucrose. We were concerned about losses of outer membrane due to this treatment, particularly in view of the known effects of EDTA (Leive, 1965; Bayer and Leive, 1977) and Tris—HCI (Irvin et a!., 1981a,b; Nogami and Mizushima, 1983) on E. colt. We undertook, therefore, a scanning electron microscope study of the effects of EDTA and Tris—HCI, as well as NaCI and sucrose and the various
f Present address: National Institute for Medical Research,TheRidgeway,Milll-IilI,LOndOn,NW7 1AA,U.K. 5
6
U. M. Lloyd. I. ap (iwynn and N. J. Rus~cll
washing procedures, on the surface morphology and cell envelope integrity of M. cri’ophi/u.s. This study has a broader relevance to the food industry since not only is M. crvophi/us a food spoilage isolate, but salt and sugar are widely used as food preservatives (Roberts and Skinner, 1983). In addition EDTA and Tris HO find clinical use in the enhancement of antibiotic efficacy (Wooley and Jones, 1983).
MATERIALS AND METHODS Bacteria and culture medium Micrococcus crvophilus (ATCC 15174) was grown at 20~Cin a Casamino acids/salts medium (Russell, 1974). Bacteria were harvested during late exponential phase of growth, when their 0D600 was approximately 0.8. Preparation of sanip!e.s for scanning electron microscopy
(i) Control, intact bacteria were deposited directly on Millipore filters (Code GS type, pore size, 0.22 j.tm) by placing a small amount of culture on the filter in a plastic holder adapted to fit tapered centrifuge tubes. These were centrifuged at 600 g for 5 mm using a bench centrifuge. The holder and contents were placed in 2~(v/v) formaldehyde in 100 mM phosphate buffer, pH 7.4 for 30 mm (ap Gwynn et a!., 1976). (ii)Treated, intact bacteria were centrifuged as above and the pellet gently resuspended in (a) 100mM phosphate buffer, pH 7.4, (b) 100 mM Tris-HCI, pH 7.5, (c) 1.3 mM EDTA, (d) 0.5 M sucrose or (e) 0.85°c,(w/v) NaCl for 1 hr before being treated as the controls above, Sphaeroplasts and isolated cytoplasmic membranes or outer membranes were prepared as described previously (Lloyd and Russell, 1984). Sphaeroplasts were placed on Millipore filters (Code GS type, pore size, 0.22 ~.tm) and treated with 2’,, (v/v) formaldehyde in 100 mM phosphate buffer, pH 7.4. for 30 mm before furlher
Li’,i~endfor
pw/t
treatment. Purified cytoplasmic and outer membrane samples were centrifuged at 143,000g (r
5,. = 6.3 cm) in a 60 Ti rotor using a Beckman L2 65B ultracentrifuge. The pellet was placed on Millipore filters (Code GS type, pore size. 0.22 l.im) and processed in the same way as the sphaeroplast samples. Subsequent steps in the preparative procedure were the same for control and treated samples. and utilized a method which minimized sample shrinkage (Wollweher ci a!.. 1981). After incubation in 2’,, formaldehyde (v/v) solutions, sampies were placed in a mixture of 2’,, (v v) formaldehyde and 2 (v/v) glutaraldehyde in 100 mM phosphate buffer, pH 7.4. for 30 miii. Further fixation was carried out in 2,, )v!v) glutaraldehyde in l0() mM phosphate buffer, pH 7.4. for 30 mm. Samples were then placed in a solution of 0s04 (1° wv) in veronal acetate buffer pH 7.2 for I hr, followed by incubation for 30mm in a saturated aqueous solution of thiocarbohydrazide followed by a further 30 mm in 0s04 to increase the binding of osmium to the samples (Kelley ci a!., 1973). The coated Millipore filters then were rinsed thoroughly with distilled water, and placed in aqueous tannic acid (1 ~, v/v) for 1 hr. Samples were then transferred to aqueous uranyl acetate (1’,, w:v) for 1 hr. Dehydration was carried out in a graded series of alcohols and finally with absolute alcohol. The sample was transferred to pure fluorisol (a fluorocarbon safety solvent: Penetone Chemicals, Bassington md. Est.. Cramlington, Northumberland) via successive washings for 10 mm each in absolute ethanol, fluorisol mixtures (3: 1. 1: I and 1:3, v,/v). The samples were dried in a Polaron E 3000 critical point dryer, and sputter coated with gold on a Polaron E 5100 series I ‘cool’ sputter coater. ,
SCanning electron microscopy
Micrographs were taken using a JEOL JEM 100 CX TEMSCAN operated in the secondary electron emission mode at 40 kV accelerating voltage.
7 Fig. I. Untreated whole cell of t-!t’t’rs’a’t’to r’rropIii/ii~.C. (‘lumped capsuk’.
Fig. 2. High power of cell surface of out rested \ 1. ~‘ri’opliilus. Note was orot u beta itcos. ~ 51)15)0.
~.
I .15K),
nato ‘c and the presence ol sniall
tlicrococeio t’rtophiius
Surface Morpholog~of ,
_I :1. V
,,,
-~
7
8
U. M. Lloyd. I. ap Gwynn and N. J. Russell
RESU LTS Micrococcus cryophi!us is a Gram-negative coccus, and in an exponentially growing culture most cells are dividing, which results in a typical ‘dumbell’ appearance of cell doublets (Mazanec et a!., 1966). The cells are covered by a loosely-bound capsular material which is easily lost during the various treatments used to prepare samples for electron microscopy (Sleytr and Kocur, 1971). This was apparent when intact, control bacteria were deposited on Millipore filters without treatment prior to initial fixation in formaldehyde. The protocol used in this study retained the capsule, but it was aggregated, thus revealing the external surface of the outer membrane (Fig. 1), which is shown at a higher magnification in Fig. 2. This figure also shows that there are small blebs (i.e. vesicular membrane evaginations) on the peaks of the outer membrane A more realisticfolds. picture of the surface structure
Table I. The comparative sizes of cells of Mü’rococrus cr.vo phi/us before and after various treatments. The sizes of the sphaeroplasts, and isolated cytoplasmic membrane yesides and outer membrane resides are also included for comparison, together with data on untreated bacteria from Mazanec ~ al. (1966)
of M. c’ryophi/us in uwo is afforded by incubation of the cells in sucrose. This treatment appears to stabilize the capsular layer, with the majority still adhering to the underlying outer membrane (Fig. 3). The surface of the capsule is relatively smooth where it has not been disrupted. It also appears to bridge the gap between the cells of fully divided doublets (Figs. 1 and 3). Incubation of M. cryophi!us in 0.85°~(w/v) NaC1 solution results in almost total removal of the capsule (Fig. 4). There is also an approximately 10°~ decrease in cell size (Table 1); this could be accounted for by loss of the capsule, which has a thickness of 0.07-~0.08~.im (Mazanec et a!., 1966). The removal of capsular material reveals the full extent of the crenellations of the outer membrane. A small amount of the outer membrane may possibly be lost, because a few small vesicles can be seen arising from the surface, but otherwise the outer membrane is intact (Fig. 4). Both 100 mM Tris—HC1 buffer, pH 7.5, and 1.3 mM EDTA remove most of the capsular material, but the clumping observed in the untreated control cells is not apparent, and there are some smooth regions of intact capsule (Figs. 5 and 8). In other respects, the actions of Tris -HCI and EDTA are quite different (Figs. 6 and 7). EDTA has a particularly dramatic effect on the outer membrane: the deep crenellations observed in the presence of Tris--HCI buffer (or sucrose) are replaced by a ‘lumpy’ appearance, and the outer membrane also forms numerous
___________________________________________
Size of cell doublet (grn) Sample,’treatment (a) Untreated cells: Mazanec Ct ~his studs’ (b) Treated cells: Sucrose NaCI Tris-l-ICI
at.
Length
Width
I II
088 08(1
110 l.l0 0.95 .00
Phosphate buffer 1.20 5 )c) Sphaeroplasts (d) Outer membrane vesidles* , (e) Cvtoplasmtc membrane residest
0.8))
0.75 0.~() 0.80 0.440 0.090 ((.06 I
Diameter Values are means of 10 measurements (except sphacroplast, S = 3). *
vesicles of approximately 50 nm dia blebbing from the surface (Fig. 7). The size of the cells treated with EDTA is smaller than of those treated with NaC1 or Tris—HC1, while those treated with sucrose are very similar to untreated cells (Table 1). As a control for the various treatments intact bacteria were incubated with 100 mM phosphate buffer, pH 7.4. The results were rather unexpected, because a quite different surface morphology was obtained (Figs. 9 and 10), compared with untreated bacteria (Figs. I and 2), or any of the other treatments (Figs. 3 -8). On incubation with phosphate buffer prior to fixation the capsule is lost completely, and the folding pattern of the outer membrane is more regular and intense (Figs. 9 and 10). This large degree of, almost regular, membrane folding increases the surface area considerably. To investigate further the nature of the cell envelope of M. cryophilus the morphology of sphaeroplasts, and of purified cytoplasmic membrane and outer membrane preparations, were examined. Sphaeroplasts lack the rigidifying peptidoglycan layer, and so tend to be fragile. and only a limited number survive the fixation procedure. Those sphaeroplasts that remain
Surface Morpholog~of .%!icroc ociu.s crt’ophiius
9
1i±w ~
-
Fig. 3. Sucrose treated ti. crtophi(us. C. Adhering capsule. ~ 80.000. Fig. 4. Nafl treated M. criophilus. Note total lack of capsule. am. Crenellated Outer membrane. x 90.000.
-
4J
10
(,
Si. Llo~d. I ap (ia \nn and N. J. Ru..’.ell
11
~
Figs 5,
it
7J
Tris—H( I treated ~l. crtophilus C. remnant of capsule being unfurled from cell surface om. Crenellated outer membrane. FigS x 45.000; Fig. 6 x 60,01)0.
Figs. 7, 8. EDTA treated M. rropliilus. C, Remnant of capsule being unfurled from cell surface; V. outer membrane vesicles being pinched off; om, knobbly (rather than wavy( outer membrane. Fig. 7 x 60.000; Fig. 8 x 45.000.
intact are spherical with a smooth surface that has several irregular vesicles attached (Fig. 11). Isolated cytoplasmic and outer membranes form vesicles which have similar surface morphologies (Figs. 12 and 13), but there is a distinct size difference—-the outer membrane vesicles are
approximately 1.5 times as large as the cytoplasmic membrane vesicles (Table I). DISCUSSION Our observations on the overall size and
9]
~io1 Figs. 9. 10. I’hosphatc treated %1. i’rvophilus. Note ver~different outer membrane niorpholog~.Fig. 9.. 30.000: Fig. lOx 90.000.
12
7
(
.
I’d Lloyd. I aI~Gis son and N .t Russell
___
4 11
I ig. II Sphacropla~isol
‘ii.
i’t’o
1’hilus my. MiLrospherulcs. Note relatisels ~mooih. ~et still liimp~membrane
so rface I ~rohahls cs topla~ni ic mciii bra tie I
lig 12 Piiritied oiiiei mcmhi,inL’
prep:ir.itioli
3. l~tiiiIiC(l e\ t~tpl:isniieiiieiiihi ,iiie
.50.ll( itt. .
pTe~’t.ii.05 ‘ii
ttt).Ottfl .
01 .1 itt)
Surface Morphology of Micrococcus cryophilus
arrangement of cells from an exponentially-growing batch culture of Micrococcus cryophilus agree with those of Mazanec et al. (1966). The majority of cells appear as doublets, because they are either dividing or have remained attached for some time after division is cornpleted. Individual cell dimensions in a doublet are 1.11 x 0.88 l.Im, whereas isolated single cells have a diameter of 0.80 )lm, and are more spherical. We did not observe giant cells (McLean et a!., 1951) or other size variations because exponentially-growing cultures were used (Mazanec et a!., 1966). All measurements must, however, be taken as being approximations to the real dimensions, as some shrinkage may well have occurred during the preparation procedure for SEM. The capsule may act as an adhesive agent because it appears to be gelatinous, judging from its appearance between cells (Fig. 3), and the manner in which it is removed by Tris—HC1 (Fig. 5) or EDTA (Figs. 7 and 8), as well as the fact that we found sloughed off capsule coating the supporting Millipore. Therefore the capsule may be important in intercellular adhesion and in retaining cell contact with the substratum; this probably also explains why cells do not separate immediately after cell division is completed. We were able to confirm by scanning electron microscopy that the outer membrane of M. cryophi/us is thrown into foldswhich gives it a wavy profile seen in transmission electron microscopy (Mazanec et a!., 1966; Lloyd and Russell, 1984), or freeze fracture electron microscopy (Sleytr and Kocur, 1971). We have demonstrated further that the outer membrane is not only folded, but has numerous small blebs arising from the peak of the folds (Fig. 2). It has been suggested that during balanced growth of Gram-negative bacteria the outer membrane is normally sloughed off and undergoes constant renewal (Irvin et a!., 1981a). Blebbing may represent the mechanism by which outer membrane is lost. Incubation of M. cryophilus in sucrose prior to fixation in formaldehyde results in the majority of the capsule being retained as a gelatinous coat (Fig. 3). In places the outer membrane can be seen through disruptions in the capsule, which gives the cell a ‘thread-bare’ appearance (Fig. 3). That only a fraction of the capsule has been removed by sucrose is confirmed by comparison with the action of NaCl, which removes most of the capsule (Fig. 4). After sucrose treatment some
13
of the deeper parts of the capsule remain in the crenellations of the outer membrane, partially veiling it—this is visualized in diagrammatic form in Fig. 14. The capsule appears to be rather gelatinous (see above). Thus during incubation in sucrose only areas which are put under stress during the subsequent fixation become detached, and the innermost parts of the capsule are trapped by the outer membrane crenellations (Fig. 14). The fact that NaCl removes these parts of the capsule indicates that the interactions between capsule and outer membrane are probably ionic. Exposure to 0.85% (w/v) NaCl did not alter the cell shape or size. This concentration is not high enough to cause the cell shrinkage seen in some other Gram-negative and Gram-positive bacteria incubated in high salt concentrations (e.g. see Johnson and Harvey, 1937; Marquis, 1968). However, there were changes in the surface morphology of M. cryophilus: the depth of folding of the outer membrane was increased and the number of blebs was reduced (Fig. 4), but it is not clear whether the former causes the latter, or
a
Ilk
..iIIuIIIIII~JHI)IIf~
capsule
outer membrane .
-
“
-
-
-
“
-
b
reia~
-
..
.,
Fig. 14. Diagrammatic representation of the removal, and partial removal, of M. crvophiius capsule.
14
U. M. Lloyd, I. ap Uwynn and N. J. Russell
whether the blebs are lost as membrane vesicles. The appearance of M. cryophilus incubated with Tris—HCI (Fig. 5) is very similar to that obtained with NaCI, except that some capsular material remains. Figure 5 shows clearly the relationship between the capsule and the outer membrane, since the remaining part of the capsule is unfurling from the outer membrane surface of the cell. There appears to be some capsule retained within the crenellations of the outer membrane producing the same kind of veiling seen with sucrose (Fig. 3) and shown diagrammatically in Fig. 14. Clearly, there are no gross morphological changes caused by Tris--HC1, most of the damage being confined to the capsule. In comparison with other bacteria Tris buffer has varied and complex effects, especially on the outer membrane of Gram-negative bacteria. For example, Nogami and Mizushima (1983) showed that not only did Tris—HCI substantially change the outer membrane morphology of E. co/i but could result in cell death, particularly in mutant strains lacking outer membrane porins. This buffer may weaken the outer membrane by disrupting ionic interactions (Schindler and Teuber, 1978), thus affecting outer membrane permeability (Irvin ci a!., 1981a,b). In some instances components of the outer membrane are released (Irvin et a!., 1981a), and the susceptibility of outer membrane proteins to chemical modification is altered (Schindler and Teuber, 1979). Similarly, EDTA drastically alters the cell walls of some Gram-negative bacteria (e.g. Roberts eta!., 1970;Moriyou and Berman, 1982). EDTA releases up to 50% of the lipopolysaccharide (Leive, 1965) and smaller amounts of lipid and protein (Leive ef a!., 1968) in certain E. co/i strains, due to disruption of the outer membrane (Bayer and Leive, 1977). This disruption also releases periplasmic enzymes (Heppel, 1972) and increases the permeability of the outer membrane to a number of antibiotics (Scudamore ci a!., 1979a,b). It was not surprising, therefore, to find that EDTA damaged the cell envelope of M. cryophi!us also; even though only l3°-~ of the outer membrane 2-keto 3-deoxyoctonate (K DO) is released by this treatment (Russell, 1972). The major effect of EDTA was on the outer membrane which lost its wavy appearance and had numerous vesicles blebbing from the surface (Fig. 7). These vesicles are larger than the blebs
seen in untreated cells (cf. Figs. 2 and 7). and probably explain the EDTA-mediated loss of outer membrane as well as the smaller cell size compared with control cells or those treated with sucrose, NaCl or Tris--HCI (Table 1). The complete loss of capsule and tight folding of the outer membrane caused by phosphate buffer (Figs. 9 and 10) was particularly surprising, since this would normally be regarded as a ‘safe’ control treatment for cells. The action of phosphate buffer on M. cryophilus is difficult to explain, and raises amoregeneral question about the importance of phosphate ions in membrane structure. Erythrocyte ghosts are more stable in the presence of phosphate buffers than in other buffers or EDTA (Pinteric ci a!., 1975). The phosphate anion may also act as a stabilizing agent on the outer membrane structure of M. cryophilus, allowing it to take on a more regular, convoluted and therefore more highly stressed conformation. The wavy pattern of the outer membrane is very regular when viewed using transmission (Mazanec ci a!., 1966; Lloyd and Russell, 1984) and freeze fracture (Sleytr and Kocur, 1971) electron microscopy. Sphaeroplasts of M. cryophilus viewed by scanning electron microscopy are spherical and have a smooth surface with a few vesicles attached (Fig. 11). This appearance differs from that seen previously by transmission electron microscopy, in which the outer membrane remains essentially intact but is separated from the cytoplasmic membrane due to cellular plasmolysis (Lloyd and Russell, 1984). In addition, the overall size of sphaeroplasts in transmission electron microscopy is approximately 2.5 times those in scanning electron microscopy. We have interpreted these differences as being due to the loss of outer membrane during the preparation procedure of scanning electron microscopy: this accounts for the size and the relatively smooth surface. The outer membrane remnants, which remain attached to the cytoplasmic membrane as vesicles, are probably at the site of the so-called ‘zones of adhesion’ where cytoplasmic and outer membranes normally make contact (Lloyd and Russell, 1984). Similar vesicles have been ohserved on the surface of yeast protoplasts when they were referred to as microspherules (Miegeville and Morin, 1977). The cause of the drastic effect on the outer membrane of sphaeroplasts in M. cryophi/us may be the critical point drying procedure or the presence of EDTA in the sphaeroplasting medium. This latter explanation
Surface Morphology of Micrococcus cryophilus
seems less likely because although EDTA does affect outer membrane morphology (Figs. 7 and 8) the membrane remains relatively intact when viewed by transmission electron microscopy (Lloyd and Russell, 1984). The reason why less of the outer membrane is lost from intact bacteria compared with sphaeroplasts is probably that the former have peptidoglycan which stabilizes the outer membrane via interactions with Braun’s lipoprotein (Lugtenberg and Van Alphen, 1983). Despite the quite dissimilar lipid and protein compositions of cytoplasmic and outer membranes of M. cryophilus (Lloyd and Russell, 1984), they form vesicles with comparative morphologies; the main difference is the larger size of the outer membrane vesicles, which confirms the results of transmission electron microscopy (Lloyd and Russell, 1984). The size difference may reflect the presence of lipopolysaccharide and the higher content of cardiolipin, and the fact that the outer membrane is more rigid (Lloyd and Russell, 1984). In conclusion, we have shown that although a number of treatments commonly used experimentally affect the structure and morphology of the cell envelope in M. cryophilus, the damage caused by chemicals like EDTA or Tris—HC1 is not as great as that reported for E. co/i. This may be due to protection by the capsule in M. cryophi/us. Also the fact that we used a wild-type strain—a number of the studies with E. co/i were only able to demonstrate outer membrane damage and loss of membrane and periplasmic components when mutants with a defective outer membrane were used (e.g. Irvin ci a!., 1981a; Nogami and Mizushima, 1983). Furthermore, there may be large variations in outer membrane stability; for example EDTA with or without Tris—HCI does not destabilize the outer membrane of Bruce/la spp. (Moriyou and Berman, 1982). Thus it is probably not warranted to generalize about the actions of these agents on all bacteria.
REFERENCES Bayer. M. E. and Leive, L.. 1977. Effect of ethylene diaminetetraacetate upon the surface ofEscherichia co/i. J. Bacteriol., 130: 1364 1381. Bohá~ek,J., Kocur, M. and Martinec, T., 1967. DNA base composition and taxonomy of some micrococci. J. gen. Microh(oI., 46: 369 375. ap Gwynn. I., Evans. P. M.. Jones. B. M. and Chandler. J. A., 1976. Shape changes and reduced calcium levels in the
15
surface membrane region of human platelets exposed to manganese in ritro. Cytobios, 16: 97 ~lO6. Heppel, L. S., 1972. The concept of periplasmic enzymes. In: Structure and Function of Biological Membranes, Rothfield, L. I (ed), Academic Press, New York, 224-247. Irvin, R. T., MacAlister, T. J., Chan, R. and Costerton, J. W., 1981a. Citrate-Tris (hydroxymethyl) aminomethane mediated release of outer membrane sections from the cell envelope a deep-rough (heptose-deficient lipopolysaccharide)ofstrain of Escherichia co/i 08. J Bacteriol., 145: 1386-1396. Irvin, R. T., MacAlister, T. J. and Costerton, J. W., 198 lb. Tris (hydroxymethyl) aminomethane buffer modification of Escherichia colt outer membrane permeability. J. Bacteriol., 145: l397—l403. Johnson, F. H. and Harvey, E. N., 1937. The osmotic and surface properties of marine luminous bacteria. J. cell. comp. Physiol., 9: 363-380. Kandler, 0., Schleifer, K. H., Niebler, E., Nakee, M., Zahradnik, H. and Reid, M., 1970. Murein types in micrococci and similar organisms. PubI. Fac. Sd. Unit’. J. E. Purkyne, Brno, K. 47: 143-156., cited in Sleytr, U. and Kocur, M., 1971. Structure of Micrococcus cryophilus after freeze etching. Arch. Mikrobiol., 78: 353-359. Kelley, R. 0., Decker, R. A. F. and Bluemink, J. G., 1973. Ligand mediated osmium binding: its application in coating biological specimens for SEM. J. Ultrastruct. Res., 45: 254-258 Leive, L., 1965. Release of lipopolysaccharide by EDTA treatment of Escherichia co/i. Biochem. Biophys. Res. Commun., 21: 290 -296. Leive, L., Shovlin, V. K. and Mergenhagen, S. E., 1968. Physical, chemical, and immunological properties oflipopolysacharide released from Escherichia co/i by ethylenediamine tetraacetate. J. biol. Chem., 243: 6384—639 1. Lloyd, U. M. and Russell, N. J., 1984. The isolation and characterisation of cryophilus. cytoplasmicCan. and J.Outer membranes from Micrococcus Microbiol., 30: 1357-1366. Lugtenberg, B. and Van Alphen, L., 1983. Molecular architecture and functioning of the outer membrane of Escherichia co/i and other Gram-negative bacteria. Biochim. biophys. Acta, 737: 51-115. Marquis, R. E., 1968. Salt-induced contraction of bacterial cell walls. J. Bacteriol., 95: 775 --781. Mazanec, K., Kocur, M. and Martinec, T., 1966. Electron microscopy of ultra-thin sections of Micrococcus cryophilus. J. Microbiol., McLean, R. Can. A., Sulzbacher, W. 12: L. 465 and-469. Mudd, S., 1951. Micrococcus crvophilus spec. nov.: a large coccus especially suitable for cytological study. J. Bacteriol., 62: 723-728. Miegeville, M. and Morin, 0., 1977. Nouvelle contribution de Ia microscopie electronique a balayage a Ser. l’etude des protoplastes de levures. C. r. Acad. Sci. Paris D., 284: 1935 1938. Moriyou. I. and Berman, D. T., 1982. Effects of nonionic, ionic, and dipolar ionic detergents and EDTA on the Bruce/la cell envelope. J. Bacteriol., 152: 822-828. Nogami,T.andMizushima,S.,1983.Outermembraneporins are important in maintenance of the surface structure of E.scherichia co/i cells. J. Bacteriol., 156: 402 408. Pinierie, L., Manery. J. F.. Chaudry, I. H. and Madapallimatiam. U., 1975. The effect of EDTA, cations and various buffers on the morphology of erythrocyte membranes: an electron microscopic siudy. Blood, 46: 709 725.
16
U. M. Lloyd. I. ap Gwynn and N. J. Russell
Roberts. N. A.. Uray, U. W. and Wilkinson, S. C.. 1970. The bactericidal action of ethylenediamine tetraacetic acid on P,seudonionas ai’ruginosa. Microh,os, 7 8: 189 208. Roberts. T. A. and Skinner, F. A., 1983. (eds.) food Microhtoloqr; idranics and Prospects. SAB Symposium No. II. Academic Press. New York. Russell, N. J., 1972. Lipids and transport in a psychrophilic bacterium. Ph.D Thesis. University of Cambridge, England. Russell. N.J., 1974. The lipid composition of the psychrophilie bacterium Micrococcu.s r-,yo ph, lii s. .1. qeti. Microhiol. 80: 217 225. Schindler, P. R. U. and Teuber, M.. 1978. Ultrastructural study of Salmonella tvphimuriuin treated with membrane— active agents: specific reaction ofdansyl chloride with cell envelope components. J. Bacteriol., 135: 198 206. Schindler, P. R. U. and Teuber, M., 1979. Fluorescent labelling of cell envelope proteins with 5-dimethyl-amino naphthalene-t-l-sulphonvl chloride-lecithin-cholesterol vesicles upon treatment of Pseudoniona,s aeruqinosa with ‘Uris (hydroxymethyl I aminomethane-hydrochloride-
cthylcnediamine tetraacetate. lEM.5 .~1icrohioI. Let!.. 6: 163 164. Scudamore. R A.. Beveridgc. 1. J. and Uoldner. M.. l979a, Penetrahi1it~of the outer membrane of Veisseria nonorrhoeae in relation to acquired resistance to penicillin and other antibiotics. in! muroh. I qenLs Chemot her.. 15: 820 827, Scudamorc, R. A.. Beseridge. ‘1. J. and Uoldner. tv., l979h. Outer membrane penetration harrier as component ol resistance to heta-laciam and other antihiotics in E,scht’ri’i’hia co/i K — 1 2. liii itnicroh. - I c/ent.~( hr’n,othcr.. IS: 182 189. Sleytr. U. and Kocur. M.. 1971. Structure of Microcorrie, crrophilu.s after frecie etching. io’h 3likrohiol . 78: 353 359 Wollwehcr. L., Straeke, R. and Uocthe, U., 1981. The use of a simple method to avoid cell shrinkage during SEM preparation i ‘i1/cro.sc..12l: 185 189. Wooley, R. E. and Jones. M. S.. 1983. Action of EDTA iris and antimicrobial agent combinations on selected pathogenie bacteria. l’~t,Microhiol., 8: 271 280.
0739-6260/85 13.00+0.00 ~ 1985 Pergamon Press Ltd.
CELL ENVELOPE STRUCTURE OF THE PSYCHROPHILIC BACTERIUM MICROCOCCUS CRYOPHILUS: A SCANNING ELECTRON MICROSCOPE STUDY GEOFFREY
M. LLOYD,*t IOLO
AP GWYNN~and NICHOLAS
J.
RUSSELI]’~
*Department of Biochemistry, University College, Cathays Park, P.O. Box 78, Cardiff, CFI 1XL, U.K. and ~Department of Zoology, University College of Wales, Penglais, Aberystwyth, Dyfed, SY23 3DA, U.K. (Received 3 October 1984)
Abstrac*—The surface morphology and structure of the cell envelope and associated capsule of the Gramnegative psychrophilic bacterium Micrococcus cryophi/us have been investigated by scanning electron microscopy, following treatment of cells with NaCI, EDTA, sucrose, Tris-HCI and phosphate buffer. These treatments removed varying amounts ofthe extracellular capsule and damaged the outer membrane to differing extents. EDTA, and to a lesserdegree Tris-HCI, caused most damage, but compared with E. co/i relatively less damage occurs, probably due to the presence of the capsule in M. cryophilus. Besides having a protective role, it is proposed that the capsule associates with the outer membrane by ionic interactions, and probably increases intercellular interaction and cell contact with the substratum. We confirm that the outer membrane has a wavy profile, and show that it is thrown into folds whose depth and regularity depend on whether Tris—HCI or phosphate buffer is used. The troughs ofthese outer membrane folds tend to retain capsular material unless cells are washed with NaCl, and the peaks of the folds form vesicles which can bleb from the surface; blebbing is greatly increased by EDTA. Index keywords: Micrococcus cryophilus, Gram-negative bacteria, cell envelope structure, psychrophilic bacteria, bacterial capsule, scanning electron microscopy.
INTRODUCTION Micrococcus cryophilus is a psychrophilic bacterium, isolated by McLean et al. (1951) from sausages made with chilled meat. Although classified originally as a Gram-positive or Gram-variable coccus, subsequent transmission electron microscopy and freeze-fracture investigations have shown it is a Gram-negative organism (Mazanec et a!., 1966; Sleytr and Kocur, 1971). Biochemical confirmation has come from studies of the guanosine and cytosine content of its DNA (Bohã~eket a!., 1967). the type of peptidoglycan in its cell wall (Kandler et a!., 1970), and from the content and composition of its lipid (Russell, 1974). Micrococcus cryophilus has a typical
Gram-negative cell wall, consisting of a cytoplasmic (‘inner’) membrane, a peptidoglycan layer, and outside that a lipopolysaccharidecontaining outer membrane. In addition there is a capsule external to the outer membrane (Mazanec et al., 1966). The cytoplasmic and outer membranes have been separated and purified, and their compositions compared (Lloyd and Russell, 1984). This purification procedure involved converting intact bacteria to sphaeroplasts using EDTA and lysozyme, Tris—HC1 buffer and sucrose. We were concerned about losses of outer membrane due to this treatment, particularly in view of the known effects of EDTA (Leive, 1965; Bayer and Leive, 1977) and Tris—HCI (Irvin et a!., 1981a,b; Nogami and Mizushima, 1983) on E. colt. We undertook, therefore, a scanning electron microscope study of the effects of EDTA and Tris—HCI, as well as NaCI and sucrose and the various
f Present address: National Institute for Medical Research,TheRidgeway,Milll-IilI,LOndOn,NW7 1AA,U.K. 5
6
U. M. Lloyd. I. ap (iwynn and N. J. Rus~cll
washing procedures, on the surface morphology and cell envelope integrity of M. cri’ophi/u.s. This study has a broader relevance to the food industry since not only is M. crvophi/us a food spoilage isolate, but salt and sugar are widely used as food preservatives (Roberts and Skinner, 1983). In addition EDTA and Tris HO find clinical use in the enhancement of antibiotic efficacy (Wooley and Jones, 1983).
MATERIALS AND METHODS Bacteria and culture medium Micrococcus crvophilus (ATCC 15174) was grown at 20~Cin a Casamino acids/salts medium (Russell, 1974). Bacteria were harvested during late exponential phase of growth, when their 0D600 was approximately 0.8. Preparation of sanip!e.s for scanning electron microscopy
(i) Control, intact bacteria were deposited directly on Millipore filters (Code GS type, pore size, 0.22 j.tm) by placing a small amount of culture on the filter in a plastic holder adapted to fit tapered centrifuge tubes. These were centrifuged at 600 g for 5 mm using a bench centrifuge. The holder and contents were placed in 2~(v/v) formaldehyde in 100 mM phosphate buffer, pH 7.4 for 30 mm (ap Gwynn et a!., 1976). (ii)Treated, intact bacteria were centrifuged as above and the pellet gently resuspended in (a) 100mM phosphate buffer, pH 7.4, (b) 100 mM Tris-HCI, pH 7.5, (c) 1.3 mM EDTA, (d) 0.5 M sucrose or (e) 0.85°c,(w/v) NaCl for 1 hr before being treated as the controls above, Sphaeroplasts and isolated cytoplasmic membranes or outer membranes were prepared as described previously (Lloyd and Russell, 1984). Sphaeroplasts were placed on Millipore filters (Code GS type, pore size, 0.22 ~.tm) and treated with 2’,, (v/v) formaldehyde in 100 mM phosphate buffer, pH 7.4. for 30 mm before furlher
Li’,i~endfor
pw/t
treatment. Purified cytoplasmic and outer membrane samples were centrifuged at 143,000g (r
5,. = 6.3 cm) in a 60 Ti rotor using a Beckman L2 65B ultracentrifuge. The pellet was placed on Millipore filters (Code GS type, pore size. 0.22 l.im) and processed in the same way as the sphaeroplast samples. Subsequent steps in the preparative procedure were the same for control and treated samples. and utilized a method which minimized sample shrinkage (Wollweher ci a!.. 1981). After incubation in 2’,, formaldehyde (v/v) solutions, sampies were placed in a mixture of 2’,, (v v) formaldehyde and 2 (v/v) glutaraldehyde in 100 mM phosphate buffer, pH 7.4. for 30 miii. Further fixation was carried out in 2,, )v!v) glutaraldehyde in l0() mM phosphate buffer, pH 7.4. for 30 mm. Samples were then placed in a solution of 0s04 (1° wv) in veronal acetate buffer pH 7.2 for I hr, followed by incubation for 30mm in a saturated aqueous solution of thiocarbohydrazide followed by a further 30 mm in 0s04 to increase the binding of osmium to the samples (Kelley ci a!., 1973). The coated Millipore filters then were rinsed thoroughly with distilled water, and placed in aqueous tannic acid (1 ~, v/v) for 1 hr. Samples were then transferred to aqueous uranyl acetate (1’,, w:v) for 1 hr. Dehydration was carried out in a graded series of alcohols and finally with absolute alcohol. The sample was transferred to pure fluorisol (a fluorocarbon safety solvent: Penetone Chemicals, Bassington md. Est.. Cramlington, Northumberland) via successive washings for 10 mm each in absolute ethanol, fluorisol mixtures (3: 1. 1: I and 1:3, v,/v). The samples were dried in a Polaron E 3000 critical point dryer, and sputter coated with gold on a Polaron E 5100 series I ‘cool’ sputter coater. ,
SCanning electron microscopy
Micrographs were taken using a JEOL JEM 100 CX TEMSCAN operated in the secondary electron emission mode at 40 kV accelerating voltage.
7 Fig. I. Untreated whole cell of t-!t’t’rs’a’t’to r’rropIii/ii~.C. (‘lumped capsuk’.
Fig. 2. High power of cell surface of out rested \ 1. ~‘ri’opliilus. Note was orot u beta itcos. ~ 51)15)0.
~.
I .15K),
nato ‘c and the presence ol sniall
tlicrococeio t’rtophiius
Surface Morpholog~of ,
_I :1. V
,,,
-~
7
8
U. M. Lloyd. I. ap Gwynn and N. J. Russell
RESU LTS Micrococcus cryophi!us is a Gram-negative coccus, and in an exponentially growing culture most cells are dividing, which results in a typical ‘dumbell’ appearance of cell doublets (Mazanec et a!., 1966). The cells are covered by a loosely-bound capsular material which is easily lost during the various treatments used to prepare samples for electron microscopy (Sleytr and Kocur, 1971). This was apparent when intact, control bacteria were deposited on Millipore filters without treatment prior to initial fixation in formaldehyde. The protocol used in this study retained the capsule, but it was aggregated, thus revealing the external surface of the outer membrane (Fig. 1), which is shown at a higher magnification in Fig. 2. This figure also shows that there are small blebs (i.e. vesicular membrane evaginations) on the peaks of the outer membrane A more realisticfolds. picture of the surface structure
Table I. The comparative sizes of cells of Mü’rococrus cr.vo phi/us before and after various treatments. The sizes of the sphaeroplasts, and isolated cytoplasmic membrane yesides and outer membrane resides are also included for comparison, together with data on untreated bacteria from Mazanec ~ al. (1966)
of M. c’ryophi/us in uwo is afforded by incubation of the cells in sucrose. This treatment appears to stabilize the capsular layer, with the majority still adhering to the underlying outer membrane (Fig. 3). The surface of the capsule is relatively smooth where it has not been disrupted. It also appears to bridge the gap between the cells of fully divided doublets (Figs. 1 and 3). Incubation of M. cryophi!us in 0.85°~(w/v) NaC1 solution results in almost total removal of the capsule (Fig. 4). There is also an approximately 10°~ decrease in cell size (Table 1); this could be accounted for by loss of the capsule, which has a thickness of 0.07-~0.08~.im (Mazanec et a!., 1966). The removal of capsular material reveals the full extent of the crenellations of the outer membrane. A small amount of the outer membrane may possibly be lost, because a few small vesicles can be seen arising from the surface, but otherwise the outer membrane is intact (Fig. 4). Both 100 mM Tris—HC1 buffer, pH 7.5, and 1.3 mM EDTA remove most of the capsular material, but the clumping observed in the untreated control cells is not apparent, and there are some smooth regions of intact capsule (Figs. 5 and 8). In other respects, the actions of Tris -HCI and EDTA are quite different (Figs. 6 and 7). EDTA has a particularly dramatic effect on the outer membrane: the deep crenellations observed in the presence of Tris--HCI buffer (or sucrose) are replaced by a ‘lumpy’ appearance, and the outer membrane also forms numerous
___________________________________________
Size of cell doublet (grn) Sample,’treatment (a) Untreated cells: Mazanec Ct ~his studs’ (b) Treated cells: Sucrose NaCI Tris-l-ICI
at.
Length
Width
I II
088 08(1
110 l.l0 0.95 .00
Phosphate buffer 1.20 5 )c) Sphaeroplasts (d) Outer membrane vesidles* , (e) Cvtoplasmtc membrane residest
0.8))
0.75 0.~() 0.80 0.440 0.090 ((.06 I
Diameter Values are means of 10 measurements (except sphacroplast, S = 3). *
vesicles of approximately 50 nm dia blebbing from the surface (Fig. 7). The size of the cells treated with EDTA is smaller than of those treated with NaC1 or Tris—HC1, while those treated with sucrose are very similar to untreated cells (Table 1). As a control for the various treatments intact bacteria were incubated with 100 mM phosphate buffer, pH 7.4. The results were rather unexpected, because a quite different surface morphology was obtained (Figs. 9 and 10), compared with untreated bacteria (Figs. I and 2), or any of the other treatments (Figs. 3 -8). On incubation with phosphate buffer prior to fixation the capsule is lost completely, and the folding pattern of the outer membrane is more regular and intense (Figs. 9 and 10). This large degree of, almost regular, membrane folding increases the surface area considerably. To investigate further the nature of the cell envelope of M. cryophilus the morphology of sphaeroplasts, and of purified cytoplasmic membrane and outer membrane preparations, were examined. Sphaeroplasts lack the rigidifying peptidoglycan layer, and so tend to be fragile. and only a limited number survive the fixation procedure. Those sphaeroplasts that remain
Surface Morpholog~of .%!icroc ociu.s crt’ophiius
9
1i±w ~
-
Fig. 3. Sucrose treated ti. crtophi(us. C. Adhering capsule. ~ 80.000. Fig. 4. Nafl treated M. criophilus. Note total lack of capsule. am. Crenellated Outer membrane. x 90.000.
-
4J
10
(,
Si. Llo~d. I ap (ia \nn and N. J. Ru..’.ell
11
~
Figs 5,
it
7J
Tris—H( I treated ~l. crtophilus C. remnant of capsule being unfurled from cell surface om. Crenellated outer membrane. FigS x 45.000; Fig. 6 x 60,01)0.
Figs. 7, 8. EDTA treated M. rropliilus. C, Remnant of capsule being unfurled from cell surface; V. outer membrane vesicles being pinched off; om, knobbly (rather than wavy( outer membrane. Fig. 7 x 60.000; Fig. 8 x 45.000.
intact are spherical with a smooth surface that has several irregular vesicles attached (Fig. 11). Isolated cytoplasmic and outer membranes form vesicles which have similar surface morphologies (Figs. 12 and 13), but there is a distinct size difference—-the outer membrane vesicles are
approximately 1.5 times as large as the cytoplasmic membrane vesicles (Table I). DISCUSSION Our observations on the overall size and
9]
~io1 Figs. 9. 10. I’hosphatc treated %1. i’rvophilus. Note ver~different outer membrane niorpholog~.Fig. 9.. 30.000: Fig. lOx 90.000.
12
7
(
.
I’d Lloyd. I aI~Gis son and N .t Russell
___
4 11
I ig. II Sphacropla~isol
‘ii.
i’t’o
1’hilus my. MiLrospherulcs. Note relatisels ~mooih. ~et still liimp~membrane
so rface I ~rohahls cs topla~ni ic mciii bra tie I
lig 12 Piiritied oiiiei mcmhi,inL’
prep:ir.itioli
3. l~tiiiIiC(l e\ t~tpl:isniieiiieiiihi ,iiie
.50.ll( itt. .
pTe~’t.ii.05 ‘ii
ttt).Ottfl .
01 .1 itt)
Surface Morphology of Micrococcus cryophilus
arrangement of cells from an exponentially-growing batch culture of Micrococcus cryophilus agree with those of Mazanec et al. (1966). The majority of cells appear as doublets, because they are either dividing or have remained attached for some time after division is cornpleted. Individual cell dimensions in a doublet are 1.11 x 0.88 l.Im, whereas isolated single cells have a diameter of 0.80 )lm, and are more spherical. We did not observe giant cells (McLean et a!., 1951) or other size variations because exponentially-growing cultures were used (Mazanec et a!., 1966). All measurements must, however, be taken as being approximations to the real dimensions, as some shrinkage may well have occurred during the preparation procedure for SEM. The capsule may act as an adhesive agent because it appears to be gelatinous, judging from its appearance between cells (Fig. 3), and the manner in which it is removed by Tris—HC1 (Fig. 5) or EDTA (Figs. 7 and 8), as well as the fact that we found sloughed off capsule coating the supporting Millipore. Therefore the capsule may be important in intercellular adhesion and in retaining cell contact with the substratum; this probably also explains why cells do not separate immediately after cell division is completed. We were able to confirm by scanning electron microscopy that the outer membrane of M. cryophi/us is thrown into foldswhich gives it a wavy profile seen in transmission electron microscopy (Mazanec et a!., 1966; Lloyd and Russell, 1984), or freeze fracture electron microscopy (Sleytr and Kocur, 1971). We have demonstrated further that the outer membrane is not only folded, but has numerous small blebs arising from the peak of the folds (Fig. 2). It has been suggested that during balanced growth of Gram-negative bacteria the outer membrane is normally sloughed off and undergoes constant renewal (Irvin et a!., 1981a). Blebbing may represent the mechanism by which outer membrane is lost. Incubation of M. cryophilus in sucrose prior to fixation in formaldehyde results in the majority of the capsule being retained as a gelatinous coat (Fig. 3). In places the outer membrane can be seen through disruptions in the capsule, which gives the cell a ‘thread-bare’ appearance (Fig. 3). That only a fraction of the capsule has been removed by sucrose is confirmed by comparison with the action of NaCl, which removes most of the capsule (Fig. 4). After sucrose treatment some
13
of the deeper parts of the capsule remain in the crenellations of the outer membrane, partially veiling it—this is visualized in diagrammatic form in Fig. 14. The capsule appears to be rather gelatinous (see above). Thus during incubation in sucrose only areas which are put under stress during the subsequent fixation become detached, and the innermost parts of the capsule are trapped by the outer membrane crenellations (Fig. 14). The fact that NaCl removes these parts of the capsule indicates that the interactions between capsule and outer membrane are probably ionic. Exposure to 0.85% (w/v) NaCl did not alter the cell shape or size. This concentration is not high enough to cause the cell shrinkage seen in some other Gram-negative and Gram-positive bacteria incubated in high salt concentrations (e.g. see Johnson and Harvey, 1937; Marquis, 1968). However, there were changes in the surface morphology of M. cryophilus: the depth of folding of the outer membrane was increased and the number of blebs was reduced (Fig. 4), but it is not clear whether the former causes the latter, or
a
Ilk
..iIIuIIIIII~JHI)IIf~
capsule
outer membrane .
-
“
-
-
-
“
-
b
reia~
-
..
.,
Fig. 14. Diagrammatic representation of the removal, and partial removal, of M. crvophiius capsule.
14
U. M. Lloyd, I. ap Uwynn and N. J. Russell
whether the blebs are lost as membrane vesicles. The appearance of M. cryophilus incubated with Tris—HCI (Fig. 5) is very similar to that obtained with NaCI, except that some capsular material remains. Figure 5 shows clearly the relationship between the capsule and the outer membrane, since the remaining part of the capsule is unfurling from the outer membrane surface of the cell. There appears to be some capsule retained within the crenellations of the outer membrane producing the same kind of veiling seen with sucrose (Fig. 3) and shown diagrammatically in Fig. 14. Clearly, there are no gross morphological changes caused by Tris--HC1, most of the damage being confined to the capsule. In comparison with other bacteria Tris buffer has varied and complex effects, especially on the outer membrane of Gram-negative bacteria. For example, Nogami and Mizushima (1983) showed that not only did Tris—HCI substantially change the outer membrane morphology of E. co/i but could result in cell death, particularly in mutant strains lacking outer membrane porins. This buffer may weaken the outer membrane by disrupting ionic interactions (Schindler and Teuber, 1978), thus affecting outer membrane permeability (Irvin ci a!., 1981a,b). In some instances components of the outer membrane are released (Irvin et a!., 1981a), and the susceptibility of outer membrane proteins to chemical modification is altered (Schindler and Teuber, 1979). Similarly, EDTA drastically alters the cell walls of some Gram-negative bacteria (e.g. Roberts eta!., 1970;Moriyou and Berman, 1982). EDTA releases up to 50% of the lipopolysaccharide (Leive, 1965) and smaller amounts of lipid and protein (Leive ef a!., 1968) in certain E. co/i strains, due to disruption of the outer membrane (Bayer and Leive, 1977). This disruption also releases periplasmic enzymes (Heppel, 1972) and increases the permeability of the outer membrane to a number of antibiotics (Scudamore ci a!., 1979a,b). It was not surprising, therefore, to find that EDTA damaged the cell envelope of M. cryophi!us also; even though only l3°-~ of the outer membrane 2-keto 3-deoxyoctonate (K DO) is released by this treatment (Russell, 1972). The major effect of EDTA was on the outer membrane which lost its wavy appearance and had numerous vesicles blebbing from the surface (Fig. 7). These vesicles are larger than the blebs
seen in untreated cells (cf. Figs. 2 and 7). and probably explain the EDTA-mediated loss of outer membrane as well as the smaller cell size compared with control cells or those treated with sucrose, NaCl or Tris--HCI (Table 1). The complete loss of capsule and tight folding of the outer membrane caused by phosphate buffer (Figs. 9 and 10) was particularly surprising, since this would normally be regarded as a ‘safe’ control treatment for cells. The action of phosphate buffer on M. cryophilus is difficult to explain, and raises amoregeneral question about the importance of phosphate ions in membrane structure. Erythrocyte ghosts are more stable in the presence of phosphate buffers than in other buffers or EDTA (Pinteric ci a!., 1975). The phosphate anion may also act as a stabilizing agent on the outer membrane structure of M. cryophilus, allowing it to take on a more regular, convoluted and therefore more highly stressed conformation. The wavy pattern of the outer membrane is very regular when viewed using transmission (Mazanec ci a!., 1966; Lloyd and Russell, 1984) and freeze fracture (Sleytr and Kocur, 1971) electron microscopy. Sphaeroplasts of M. cryophilus viewed by scanning electron microscopy are spherical and have a smooth surface with a few vesicles attached (Fig. 11). This appearance differs from that seen previously by transmission electron microscopy, in which the outer membrane remains essentially intact but is separated from the cytoplasmic membrane due to cellular plasmolysis (Lloyd and Russell, 1984). In addition, the overall size of sphaeroplasts in transmission electron microscopy is approximately 2.5 times those in scanning electron microscopy. We have interpreted these differences as being due to the loss of outer membrane during the preparation procedure of scanning electron microscopy: this accounts for the size and the relatively smooth surface. The outer membrane remnants, which remain attached to the cytoplasmic membrane as vesicles, are probably at the site of the so-called ‘zones of adhesion’ where cytoplasmic and outer membranes normally make contact (Lloyd and Russell, 1984). Similar vesicles have been ohserved on the surface of yeast protoplasts when they were referred to as microspherules (Miegeville and Morin, 1977). The cause of the drastic effect on the outer membrane of sphaeroplasts in M. cryophi/us may be the critical point drying procedure or the presence of EDTA in the sphaeroplasting medium. This latter explanation
Surface Morphology of Micrococcus cryophilus
seems less likely because although EDTA does affect outer membrane morphology (Figs. 7 and 8) the membrane remains relatively intact when viewed by transmission electron microscopy (Lloyd and Russell, 1984). The reason why less of the outer membrane is lost from intact bacteria compared with sphaeroplasts is probably that the former have peptidoglycan which stabilizes the outer membrane via interactions with Braun’s lipoprotein (Lugtenberg and Van Alphen, 1983). Despite the quite dissimilar lipid and protein compositions of cytoplasmic and outer membranes of M. cryophilus (Lloyd and Russell, 1984), they form vesicles with comparative morphologies; the main difference is the larger size of the outer membrane vesicles, which confirms the results of transmission electron microscopy (Lloyd and Russell, 1984). The size difference may reflect the presence of lipopolysaccharide and the higher content of cardiolipin, and the fact that the outer membrane is more rigid (Lloyd and Russell, 1984). In conclusion, we have shown that although a number of treatments commonly used experimentally affect the structure and morphology of the cell envelope in M. cryophilus, the damage caused by chemicals like EDTA or Tris—HC1 is not as great as that reported for E. co/i. This may be due to protection by the capsule in M. cryophi/us. Also the fact that we used a wild-type strain—a number of the studies with E. co/i were only able to demonstrate outer membrane damage and loss of membrane and periplasmic components when mutants with a defective outer membrane were used (e.g. Irvin ci a!., 1981a; Nogami and Mizushima, 1983). Furthermore, there may be large variations in outer membrane stability; for example EDTA with or without Tris—HCI does not destabilize the outer membrane of Bruce/la spp. (Moriyou and Berman, 1982). Thus it is probably not warranted to generalize about the actions of these agents on all bacteria.
REFERENCES Bayer. M. E. and Leive, L.. 1977. Effect of ethylene diaminetetraacetate upon the surface ofEscherichia co/i. J. Bacteriol., 130: 1364 1381. Bohá~ek,J., Kocur, M. and Martinec, T., 1967. DNA base composition and taxonomy of some micrococci. J. gen. Microh(oI., 46: 369 375. ap Gwynn. I., Evans. P. M.. Jones. B. M. and Chandler. J. A., 1976. Shape changes and reduced calcium levels in the
15
surface membrane region of human platelets exposed to manganese in ritro. Cytobios, 16: 97 ~lO6. Heppel, L. S., 1972. The concept of periplasmic enzymes. In: Structure and Function of Biological Membranes, Rothfield, L. I (ed), Academic Press, New York, 224-247. Irvin, R. T., MacAlister, T. J., Chan, R. and Costerton, J. W., 1981a. Citrate-Tris (hydroxymethyl) aminomethane mediated release of outer membrane sections from the cell envelope a deep-rough (heptose-deficient lipopolysaccharide)ofstrain of Escherichia co/i 08. J Bacteriol., 145: 1386-1396. Irvin, R. T., MacAlister, T. J. and Costerton, J. W., 198 lb. Tris (hydroxymethyl) aminomethane buffer modification of Escherichia colt outer membrane permeability. J. Bacteriol., 145: l397—l403. Johnson, F. H. and Harvey, E. N., 1937. The osmotic and surface properties of marine luminous bacteria. J. cell. comp. Physiol., 9: 363-380. Kandler, 0., Schleifer, K. H., Niebler, E., Nakee, M., Zahradnik, H. and Reid, M., 1970. Murein types in micrococci and similar organisms. PubI. Fac. Sd. Unit’. J. E. Purkyne, Brno, K. 47: 143-156., cited in Sleytr, U. and Kocur, M., 1971. Structure of Micrococcus cryophilus after freeze etching. Arch. Mikrobiol., 78: 353-359. Kelley, R. 0., Decker, R. A. F. and Bluemink, J. G., 1973. Ligand mediated osmium binding: its application in coating biological specimens for SEM. J. Ultrastruct. Res., 45: 254-258 Leive, L., 1965. Release of lipopolysaccharide by EDTA treatment of Escherichia co/i. Biochem. Biophys. Res. Commun., 21: 290 -296. Leive, L., Shovlin, V. K. and Mergenhagen, S. E., 1968. Physical, chemical, and immunological properties oflipopolysacharide released from Escherichia co/i by ethylenediamine tetraacetate. J. biol. Chem., 243: 6384—639 1. Lloyd, U. M. and Russell, N. J., 1984. The isolation and characterisation of cryophilus. cytoplasmicCan. and J.Outer membranes from Micrococcus Microbiol., 30: 1357-1366. Lugtenberg, B. and Van Alphen, L., 1983. Molecular architecture and functioning of the outer membrane of Escherichia co/i and other Gram-negative bacteria. Biochim. biophys. Acta, 737: 51-115. Marquis, R. E., 1968. Salt-induced contraction of bacterial cell walls. J. Bacteriol., 95: 775 --781. Mazanec, K., Kocur, M. and Martinec, T., 1966. Electron microscopy of ultra-thin sections of Micrococcus cryophilus. J. Microbiol., McLean, R. Can. A., Sulzbacher, W. 12: L. 465 and-469. Mudd, S., 1951. Micrococcus crvophilus spec. nov.: a large coccus especially suitable for cytological study. J. Bacteriol., 62: 723-728. Miegeville, M. and Morin, 0., 1977. Nouvelle contribution de Ia microscopie electronique a balayage a Ser. l’etude des protoplastes de levures. C. r. Acad. Sci. Paris D., 284: 1935 1938. Moriyou. I. and Berman, D. T., 1982. Effects of nonionic, ionic, and dipolar ionic detergents and EDTA on the Bruce/la cell envelope. J. Bacteriol., 152: 822-828. Nogami,T.andMizushima,S.,1983.Outermembraneporins are important in maintenance of the surface structure of E.scherichia co/i cells. J. Bacteriol., 156: 402 408. Pinierie, L., Manery. J. F.. Chaudry, I. H. and Madapallimatiam. U., 1975. The effect of EDTA, cations and various buffers on the morphology of erythrocyte membranes: an electron microscopic siudy. Blood, 46: 709 725.
16
U. M. Lloyd. I. ap Gwynn and N. J. Russell
Roberts. N. A.. Uray, U. W. and Wilkinson, S. C.. 1970. The bactericidal action of ethylenediamine tetraacetic acid on P,seudonionas ai’ruginosa. Microh,os, 7 8: 189 208. Roberts. T. A. and Skinner, F. A., 1983. (eds.) food Microhtoloqr; idranics and Prospects. SAB Symposium No. II. Academic Press. New York. Russell, N. J., 1972. Lipids and transport in a psychrophilic bacterium. Ph.D Thesis. University of Cambridge, England. Russell. N.J., 1974. The lipid composition of the psychrophilie bacterium Micrococcu.s r-,yo ph, lii s. .1. qeti. Microhiol. 80: 217 225. Schindler, P. R. U. and Teuber, M.. 1978. Ultrastructural study of Salmonella tvphimuriuin treated with membrane— active agents: specific reaction ofdansyl chloride with cell envelope components. J. Bacteriol., 135: 198 206. Schindler, P. R. U. and Teuber, M., 1979. Fluorescent labelling of cell envelope proteins with 5-dimethyl-amino naphthalene-t-l-sulphonvl chloride-lecithin-cholesterol vesicles upon treatment of Pseudoniona,s aeruqinosa with ‘Uris (hydroxymethyl I aminomethane-hydrochloride-
cthylcnediamine tetraacetate. lEM.5 .~1icrohioI. Let!.. 6: 163 164. Scudamore. R A.. Beveridgc. 1. J. and Uoldner. M.. l979a, Penetrahi1it~of the outer membrane of Veisseria nonorrhoeae in relation to acquired resistance to penicillin and other antibiotics. in! muroh. I qenLs Chemot her.. 15: 820 827, Scudamorc, R. A.. Beseridge. ‘1. J. and Uoldner. tv., l979h. Outer membrane penetration harrier as component ol resistance to heta-laciam and other antihiotics in E,scht’ri’i’hia co/i K — 1 2. liii itnicroh. - I c/ent.~( hr’n,othcr.. IS: 182 189. Sleytr. U. and Kocur. M.. 1971. Structure of Microcorrie, crrophilu.s after frecie etching. io’h 3likrohiol . 78: 353 359 Wollwehcr. L., Straeke, R. and Uocthe, U., 1981. The use of a simple method to avoid cell shrinkage during SEM preparation i ‘i1/cro.sc..12l: 185 189. Wooley, R. E. and Jones. M. S.. 1983. Action of EDTA iris and antimicrobial agent combinations on selected pathogenie bacteria. l’~t,Microhiol., 8: 271 280.